Inflammasomes alert the mammalian immune system to the presence of infection and tissue damage. These sensory complexes activate caspase-1 in the canonical pathway to cleave IL-1 family cytokines for secretion and eliminate infected host cells via a form of lytic cell death termed pyroptosis. In addition, a new non- canonical inflammasome has emerged that enlists caspase-11 specifically in response to Gram-negative bacteria. How each pathway discriminates microbial from danger signals and the host proteins responsible for this functional dichotomy remain unknown. Both questions are central to understanding the biology of inflammasome-mediated host defense. Here, we tackle this issue by focusing on a new family of 65-73kDa immune GTPases termed the Guanylate binding Proteins (GBPs) that uncouple these two activities. GBP5 specifically promotes canonical NLRP3 inflammasome activation to microbial but not danger signals, the first such protein to exhibit this profile. Subsequent work has found GBP1 and GBP2 confer the same selectivity. Thus the GBPs offer a unique opportunity to understand how one side of this bimodal response operates. In Aim 1 we will test whether GBP1, GBP2 and GBP5 promotes not only the canonical inflammasome pathway but also the non-canonical inflammasome triggered by Gram-negative bacterial stimuli. This will be examined using genetically-deficient human and mouse macrophages as well as newly-created Gbp1-/-, Gbp2-/- and Gbp5-/- mice infected with Salmonella typhimurium (Stm) variants that stimulate either inflammasome pathway. Our powerful genetic approach will reveal which GBP governs what inflammasome activities in vitro and in vivo. Thereafter, we will dissect the molecular mechanisms used by the inflammasome-related GBPs to confer their intracellular functions as part of Aim 2. Here gene-deficient macrophages complemented with GBP mutants with distinct biochemical lesions will test whether they are involved in cytosolic ligand detection or spatial assembly of the inflammasome complex during infection. Reconstitution studies will also reveal if the core inflammasome machinery is physically assembled by the GBPs as part of this macromolecular complex. Collectively, our proposal examines a new set of host factors that partition the inflammasome response with important implications for human therapies designed to promote antimicrobial activities while avoiding tissue damage.